G-protein-coupled receptors (GPCRs) represent the largest group of targets for drug discovery. Drug development for these receptors has relied heavily on high-throughput screening of large libraries of compounds. This strategy has seen some successes for a number of receptors, particularly Class A (rhodopsin family) GPCRs for non-peptide hormones and neurotransmitters. However, this approach has been mostly unsuccessful for Class B GPCRs. In this subfamily, the endogenous ligands are all relatively large peptides ranging in size from 27 to 84 amino acids. Class B GPCRs are characterized by an extended ligand recognition surface that includes residues from the transmembrane domains, from the large N- terminal domain, and from the interhelical extracellular segments. This extended ligand recognition surface likely explains the difficulty in developing small molecule drugs selective for Class B GPCRs. Alternative or complementary approaches thus need to be developed that include high-resolution structural information, which can in turn be used to design drugs based on the receptor structure. At present, only a handful of GPCR structures have been solved, and none within the Class B GPCR subfamily. A recent advance in crystallization technology, generating GPCR-T4 Lysozyme (GPCR-T4L) fusion proteins, has led to high-resolution structures of two Class A GPCRs, opening new opportunities for structure-based design of drugs. ConfometRx has obtained the exclusive licensing rights for the commercial application of GPCR-T4L technology, which was developed in Brian Kobilka's lab at Stanford University. We propose here to adapt this technology to Class B receptors. In phase I of this SBIR, we aim to determine the feasibility of using the GPCR-T4L technology to express and purify a functional T4L fusion receptor for a selected Class B GPCR. In phase II, we will proceed with the crystallization trials and structure determination. In addition, once the methodology developed here is established, it will be applied to other Class B GPCRs. The structural information collected with this approach could become a critical component for the successful development of new small molecule drugs. PUBLIC HEALTH RELEVANCE: In this SBIR, we propose to determine the three-dimensional structure of a receptor that belongs to an important family of drug targets, the Class B family of G-protein-coupled receptors. They participate in the physiological regulation of metabolism and growth, and in immuno/neuro-modulations as well as endocrine/exocrine secretions, but drug development targeted at these receptors has been mostly unsuccessful. By solving the structure of such a receptor, we hope to provide a critical tool for the development of new drugs.